Automated compensation of light attenuation in confocal microscopy by exact histogram specification

Confocal laser scanning microscopy (CLSM) enables us to capture images representing optical sections on the volume of a specimen. The images acquired from different layers have a different contrast: the images obtained from the deeper layers of the specimen will have a lower contrast with respect to the images obtained from the topmost layers. The main reasons responsible for the effects described above are light absorption and scattering by the atoms and molecules contained in the volume through which the light passes. Also light attenuation can be caused by the inclination of the observed surface. In the case of the surfaces that have a steep inclination, the reflected light will have a different direction than the one of the detector. We propose a technique of digital image processing that can be used to compensate the effects of light attenuation based on histogram operations. We process the image series obtained by CLSM by exact histogram specification and equalization. In this case, a strict ordering among pixels must be induced in order to achieve the exact histogram modeling. The processed images will end up having exactly the specified histogram and not a histogram with a shape that just resembles to the specified one, as in the case of classical histogram specification algorithms. Experimental results and theoretical aspects of the induced ordering are discussed, as well as a comparison between several histogram modeling techniques with respect to the processing of image series obtained by confocal microscopy.